The overall goal of this renewal continues to be the study of the integrin ?6?4 (referred to as `?4') as an approach for elucidating mechanisms involved in the genesis and progression of breast carcinoma, and for identifying targets for the clinical management of this disease. This integrin anchors basal epithelial cells to the basement membrane in inert structures termed hemidesmosomes (HDs). A seminal finding made in work funded by this grant is that ?4 is mobilized from HDs in invasive carcinomas and it translocates to the leading edge of cells where it engages F-actin and promotes migration/invasion. Elucidating the mechanism that regulates this mobilization of ?4 is essential for understanding its contribution to carcinoma biology. Also, much more needs to be learned about ?4 expression and function in human breast cancer. Indeed, a significant observation made with current funding is that ?4 expression correlates with basal-like tumors, aggressive tumors that retain features of basal epithelial cells and lack expression of ER, PR and HER2 (`triple negative'). Moreover, a `?4 gene signature'has been generated, a cluster of 90 genes whose expression correlates significantly with ?4 in human breast tumors and that is prognostic for reduced survival and tumor recurrence. A new phase in the study of ?4 and cancer will be initiated based on the hypothesis that ?4 functions in concert with multiple proteins to drive a specific type of breast tumor and that ?4 is a powerful tool for understanding the contribution of these other proteins to breast cancer. The first aim will assess the hypothesis that PKC-?-mediated phosphorylation of three serine residues (S1356, S1360, S1364) in the ?4 intracellular domain triggers the mobilization of ?4 from HDs to F-actin and enables it to function in migration and invasion, and as a signaling receptor. This aim will be accomplished by generating a transgenic `knock-in'mouse in which these serines are mutated to alanines. Wound healing, ?4 signaling, and the genesis and progression of basal-like tumors will be assessed in these mice. The second aim will define the relationship between ?4 and actin-binding proteins that are members of the ?4 signature. Specifically, the actin-bundling protein fascin will be studied because its expression correlates with basal-like tumors and its functions could facilitate tumor progression. The contribution of fascin to the progression of breast tumors will be determined, and the hypothesis that ?4 regulates fascin localization in actin protrusions and that fascin is necessary for ?4-dependent migration and invasion will be assessed. The hypothesis that PKC-? regulates ?4 and fascin coordinately to drive migration and invasion will also be examined. The third aim focuses on SOX9, a transcription factor that is in the ?4 signature and the original basal-like gene cluster but whose functions in breast are unknown. The contribution of SOX9 to mammary gland development will be determined by generating a Sox9 targeted deletion in this epithelium. The involvement of SOX9 in basal-like tumors and its relationship to the ?4 signature will also be assessed. This proposal seeks to understand a highly aggressive form of breast cancer termed `basal-like'that is common among women with mutations in BRCA1 and in pre-menopausal African-American women. We have identified several basal-like genes whose expression is linked to increased tumor recurrence and decreased survival in women with breast cancer. The goals of this proposal are to understand how the proteins encoded by these genes contribute to breast cancer and how their functions are inter-related.